Following a meal, increased blood glucose levels stimulate insulin release from the pancreas to act throughout the body to lower blood glucose levels. Important sites of action of insulin on glucose metabolism include facilitation of glucose uptake into skeletal muscle and adipocytes, and an increase of glycogen storage in the liver. Skeletal muscle and adipocytes is responsible for insulin-mediated glucose uptake and utilization in the fed state, making them very important site for glucose metabolism.
Diabetes comprises two distinct diseases, type 1 (or insulin-dependent diabetes) and type 2 (insulin-independent diabetes), both of which involve the malfunction of glucose homeostasis. Type 2 diabetes affects more than 350 million people in the world and the number is rising rapidly. Complications of diabetes include severe cardiovascular problems, kidney failure, peripheral neuropathy, blindness and even loss of limbs and death in the later stages of the disease. Type 2 diabetes is characterized by insulin resistance in skeletal muscle and adipose tissue (fat), and at present there is no definitive treatment. Most treatments used today are focused treating dysfunctional insulin signaling or inhibiting glucose output from the liver and many of those treatments have several drawbacks and side effects. There is thus a great interest in identifying novel insulin-independent ways to treat different form of metabolic orders connected with dysregulation of glucose uptake such as type 2 diabetes.
In type 2 diabetes the insulin-signaling pathway is blunted in peripheral tissues such as fat and skeletal muscle. Methods for treating type 2 diabetes typically include lifestyle changes, as well as the administration of insulin or oral medications to help the body with the glucose homeostasis. People with type 2 diabetes in the later stages of the disease develop “beta-cell failure” or the inability of the pancreas to release insulin in response to high blood glucose levels. In the later stages of the disease patients often require insulin injections, in combination with oral medications, to manage their diabetes. In type 2 diabetes the insulin-signaling pathway is blunted in peripheral tissues. Furthermore, most common drugs have side effects including the said downregulation or desensitization of the insulin pathway and/or the promotion of fat incorporation in fat, liver and skeletal muscle, as well as increased stimulation of proliferation of certain cells and a higher risk of promoting cancer. There is thus a great interest in identifying novel ways to treat metabolic diseases including type 2 diabetes that do not include these said side-effects.
The molecular understanding of the signaling pathway below the insulin receptor has been a very hard problem to solve and have been occupying a great number of researchers since the discovery of insulin. In short, control of glucose uptake by insulin involves activation of the insulin receptor (IR), insulin receptor substrate (IRS), phosphoinositide 3-kinase (PI3K) and thus stimulation of phosphatidylinositol (3,4,5)-triphosphate (PIP3), mammalian target of rapamycin also called mechanistic target of rapamycin (mTOR), Akt/PKB (Akt) and TBC1D4 (AS160), leading to translocation of glucose transporter 4 (GLUT4). Akt activation is considered necessary for GLUT4 translocation.
It should be noted that skeletal muscles make up a major part of mammals and have a vital role in the regulation of systemic glucose metabolism, being responsible for up to 85% of whole-body glucose disposal (DeFronzo et al. 1981). Glucose uptake in skeletal muscles is regulated by several intra- and extracellular signals. The hormone insulin is the most well studied of the signals but other signals also exist. For example, AMP activated kinase, AMPK, functions as an energy sensor in the cell, which can increase glucose uptake and fatty acid oxidation. Also muscle contraction in itself can cause increased glucose uptake. Due to the great influence skeletal muscles have on glucose homeostasis it likely that further mechanisms exists. In the light of the increases prevalence of type II diabetes, it is of great interest to find and characterize novel insulin-independent mechanisms to increase glucose uptake in muscle cells.
Insulin and catecholamines are released in the body in response to quite different stimuli. Whereas insulin is released in response to the rise in blood sugar levels after a meal, epinephrine and norepinephrine are released due to various internal and external stimuli, such as exercise, emotions and stress but also homeostatic tissue regulation. Insulin is an anabolic hormone that stimulates many processes involved in growth including glucose uptake, glycogen and triglyceride formation whereas catecholamines are mainly catabolic. Although insulin and catecholamines normally have antagonistic effects, we have shown previously that they have similar actions in skeletal muscle on glucose uptake (Nevzorova et al. 2002). It is likely that catecholamines stimulate glucose uptake via adrenergic receptors (Nevzorova et al. 2006, Hutchinson, Bengtsson 2005) which are prototypical models for G protein-coupled receptors (GPCRs) and their signaling (Santulli, Iaccarino 2013, Drake, Shenoy & Lefkowitz 2006) to supply muscle cells with an energy substrate. Thus it is likely that in mammals, including humans, that GPCRs and insulin systems can work independently to provide for the energy need of skeletal muscle during different situations. Since insulin stimulate many anabolic processes including a number of unwanted side effects it would be beneficial to be able to stimulate glucose uptake through GPCRs that does not include many of the unwanted processes in the insulin signaling pathway.
It is well known in the art that adrenergic receptors are prototypical models for the study of G protein-coupled receptors (GPCRs) and their signaling (Santulli, Iaccarino 2013, Drake, Shenoy & Lefkowitz 2006). There are three different classes of ARs, with distinct expression pattern and pharmacological profiles: α1-, α2- and β-ARs. The α1-ARs comprise the α1A, α1B and α1D while α2-ARs are divided into α2A, α2B and α2C. The β-ARs are also divided into the subtypes β1, β2, and β3, of which β2-AR is the major isoform in skeletal muscle cells (Watson-Wright, Wilkinson 1986, Liggett, Shah & Cryer 1988). Adrenergic receptors are G protein coupled and signal through classical secondary messengers such as cAMP and phospholipase C and are thus suited as prototypical models for most classes of GPCRs. GPCRs are expressed in various tissues and many effects occurring downstream of GPCRs in skeletal muscles has been attributed to classical secondary messenger signaling, but there are also atypical events downstream of GPCRs dependent on a protein family called G protein-coupled receptor kinases (GRKs), and these are kinases which can phosphorylate the intracellular loops as well as the c-terminal tail of the receptor when it is activated. The different GRK isoforms give a distinct phosphorylation pattern, thus directing the further signal. This can be desensitization of the signal, internalization of the receptors and recruitment of β-arrestins. GRKs phosphorylate GPCRs domains after the GPCR has been activated resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent way via direct protein-protein interaction. In short GRKs phosphorylate serine and threonine residues on the GPCR in the intracellular domains which act as a docking site for proteins for example arrestins that are involved in desensitization of the GPCRs but as mentioned GRKs also regulate cellular responses independent on their kinase activity. Emerging evidence suggests that in particular GRK2 interacts with a diverse number of non-GPCR substrates (Evron, Daigle & Caron 2012a) modulating multiple cellular responses in various physiological contexts.
It has have previously been shown that GPCR and GRKs can increase glucose uptake in Chinese hamster ovary (CHO) cells (Dehvari et al. 2011) but this cell-line has little or no relevance to murine or human cells involved in glucose homeostasis in vivo. It has thus been unclear if GRKs can stimulate glucose uptake in relevant cells for glucose homeostasis and, if so, through which mechanisms.
GPCR stimulated glucose uptake has been attributed to classical secondary messenger stimulation such as increase in cAMP levels, phospholipase C (PLC) activity and calcium levels (Gilman 1987). The increase of these classical secondary messengers has many effects in different tissues. For example, it increases heart rate, regulates blood flow, airflow in lungs and increases release of glucose from the liver, which all can be detrimental or be considered unwanted side effects if stimulation of GPCRs should be considered as a diabetes treatment. Adverse effects of GPCR agonists are for example tachycardia, palpitation, tremor, sweats, agitation and increased glucose levels in the blood (glucose output from the liver). All these effects can be attributed to GPCR stimulated elevation of classical secondary messengers in various tissues. It would thus be beneficial to be able to activate GPCR without activating these classical secondary messengers to increase glucose uptake in peripheral tissues without stimulating the unwanted side effects.
Glucose uptake is mainly stimulated via facilitative glucose transporters (GLUT) that mediate glucose uptake into most cells. GLUTs are transporter proteins that mediate transport of glucose and/or fructose over the plasma membrane down the concentration gradient. There are fourteen known members of the GLUT family, named GLUT1-14, divided into three classes (Class I, Class II and Class III) dependent on their substrate specificity and tissue expression. GLUT1 and GLUT4 are the most intensively studied isoforms and, together with GLUT2 and GLUT3, belong to Class I which mainly transports glucose (in contrast to Class II that also transports fructose). GLUT1 is ubiquitously expressed and is responsible for basal glucose transport. GLUT4 is only expressed in peripheral tissues such as skeletal muscle, cardiac muscle and adipose tissues. GLUT4 has also been reported to be expressed in e.g. brain, kidney, and liver. GLUT4 is the major isoform involved in insulin stimulated glucose uptake. To treat a condition involving a dysregulation of glucose homeostasis or glucose uptake in a mammal, it is of paramount importance to activate certain GLUTs. For example for diseases such as type 2 diabetes it is vital to activate GLUT4 translocation to the plasma membrane and thus glucose uptake. Regulation of GLUT1 translocation or intrinsic activity has been suggested to occur in several tissues including erythrocytes depending on ATP-levels (Hebert, Carruthers 1986). It has also been indicated in HEK-cells (Palmada et al. 2006), 3T3-L1 (Harrison et al. 1992) and clone-9 cells (Barnes et al. 2002). Impaired GLUT translocation, of in particular GLUT8, has been reported as involved in both male and female infertility (Gawlik et al. 2008, Carayannopoulos et al. 2000). The mechanism whereby insulin signaling increases glucose uptake is mainly via GLUT4-translocation from intracellular storage to the plasma membrane (Rodnick et al. 1992). After longer insulin stimulation also GLUT1-content is increased due to increased transcription (Taha et al. 1995). Glucose uptake in type 2 diabetes is associated with defects in PI3K activity, insulin receptor tyrosine, IRS and Akt phosphorylation, resulting in impairment of GLUT4 translocation to the plasma membrane. Impaired GLUT translocation also plays a role in muscle wasting. Furthermore, GLUT translocation plays a role in feeding behavior. Mice lacking GLUT4 develop problems with lipid and glucose homeostasis leading to changes in feeding behavior. Decreased concentrations of GLUT1 and GLUT3 have also been shown in the brains of patients with Alzheimer's disease (Simpson et al. 2008). Also in a review article of Shah K, et al. (Shah, Desilva & Abbruscato 2012) the role of glucose transporters in brain disease, diabetes and Alzheimer's disease is discussed.